Endothelial cell apoptosis is an important means of regulating angiogenesis and may play a role in the pathogenesis of diseases involving the alveolar capillary septum. Small GTPases possess a C-terminal CAAX motif and undergo post-translational processing, culminating in carboxyl methylation of C-terminal cysteine by isoprenylcysteine carboxyl methyltransferase(ICMT). Based on Preliminary Results, we hypothesize that inhibition of ICMT decreases carboxyl methylation of Ras and RhoA GTPases, and causes disruption of Focal Adhesion Complexes (FAC), caspase activation, proteolysis of FAC protein components, and apoptosis. Preliminary Results indicate that ICMT inhibition changes expression and charge of GRP94, a chaperone protein that is important in the Unfolded Protein Response (UPR). Since malfunction of the UPR and endoplasmic reticulum (ER) stress response causes apoptosis, we hypothesize that decreased ICMT activity and resulting decreased Ras or RhoA activity alter GRP94 function, resulting in apoptosis due to malfunction of the UPR. 1. We will determine the effects of ICMT inhibition on FAC formation and anoikis. a. We will determine the effects of inhibitors of ICMT on methylation, localization, and activation of Ras and RhoA GTPase and on apoptosis using cultured pulmonary vascular endothelial cells, b. We will determine the role of Ras and RhoA GTPases in endothelial anoikis caused by ICMT inhibition by comparing the effects of over-expression of Ras or RhoA GTPase and downstream signaling molecules on FAC disruption and apoptosis caused by ICMT inhibition, c. We will determine the effects of ICMT inhibition on pulmonary vascular endothelial apoptosis in vivo. 2. We will determine the effect of ICMT inhibition on GRP94 and the role of the ER Stress Response in endothelial cell apoptosis caused by ICMT inhibition, a. We will determine the effects of ICMT inhibition on GRP94 expression, post-translational processing, and sub-cellular localization. b.We will determine the role of decreased small GTPase activity by assessing effects of Ras and RhoA over-expression on ICMT-induced changes in GRP94. c.We will determine the effects of ICMT inhibition on markers of the UPR/ER Stress Response. d.We will determine the effects of Ras and/or RhoA over-expression on the UPR/ER Stress Response. e.We will determine the effects of GRP94 over-expression on caspase activation, FAC disruption, proteolysis of FAC components, and anoikis induced by inhibition of ICMT. The work proposed in this application will ascertain the mechanism of GTPase methylation and the role of GRP94 in regulation of endothelial cell apoptosis. This work will improve understanding of lung diseases characterized by endothelial apoptosis, such as emphysema. In addition, understanding of apoptosis may provide clues to treatment of lung diseases dependent upon abnormal endothelial proliferation, such as Pulmonary Arterial Hypertension and cancer.